During the past grant cycle, using a variety of rat models, we have proven that Rab38 is responsible for the quantitative trait locus (QTL) named Rf-2, identified and validated that Sorcs1 is responsible for the QTL named Rf-1, and have identified that Shroom3 causes changes in glomerular permeability. We have made considerable strides towards cloning by position two other QTL, Rf-3 and Rf-4. In collaboration with the Chronic Kidney Disease Genetics (CKDGen) Consortium, we found that SORCS1 and SHROOM3 are significantly associated with renal disease in humans, demonstrating that this work has clinical relevance. Our long-term goal is to define the genetic architecture of end stage renal disease (ESRD). Our data suggests that a defect in both the glomerular permeability and protein trafficking in the proximal tubules is required for proteinuria, which is now our centrl hypothesis. The proposed Specific Aims are to 1) test a collection of human sequence variants of SHROOM3 predicted to be dysfunctional; 2) identify the genes/genomic elements underlying the QTL Rf-3 (interval size 1.81 Mb) and QTL Rf-4 (interval size 0.9 Mb) participating in glomerular permeability, which is thought to initiate proteinuria; and 3) test the central hypothesis that dysfunction in both glomerular permeability and protein trafficking in the proximal tubules are participating together in the development of proteinuria. Testing this central hypothesis requires the use of whole animals and cannot be done in human. Our genetic/genomic infrastructure for this proposal includes: complete genomic sequence of parental strains, NextGen sequencing tools including bioinformatic analysis, narrow congenic strains, three identified causative genes, as well as detailed physiological characterization creating an ideal platform for discovering important new genes and testing this new central hypothesis. We will study human variants of SHROOM3 in HEK293 cells, zebrafish and humanized sensitized FHH rat model for CKD. Specifically, Aim 1 tests a collection of human variants for renal disease and demonstrates how model systems can be used to follow-up genes nominated by many genome-wide association studies (GWAS). Our collaboration with the CKDGen enables us to test if the genes identified for Rf-3 and Rf-4 contribute to human disease; thereby, providing knowledge about the disease process in humans. The rationale for the proposed research is the test of human variation in SHROOM3, identification of new genes, and a new hypothesis which will provide new insights into this disease process and could identify new targets for future treatment of renal disease with a rising incidence and limited effective treatments. Specific aims 1-3 are expected to reveal the knowledge about how mutated SHROOM3 functions and identify two new genes, which will either link to known pathways or unmask new ones. We will also determine if two hits (one in the glomeruli and one in proximal tubules) are involved. Each aim of this proposal will provide fundamental advances toward defining the genetic architecture of proteinuria which is often the precursor of ESRD.